.alpha.-Amylases (.alpha.-1,4-glucan-4-glucanohydrolase, EC 3.2.1.1) hydrolyze internal .alpha.-1,4-glucosidic linkages in starch, largely at random, to produce smaller molecular weight malto-dextrins. .alpha.-Amylases are of considerable commercial value, being used in the initial stages (liquefaction) of starch processing; in alcohol production; as cleaning agents in detergent matrices; and in the textile industry for starch desizing. .alpha.-Amylases are produced by a wide variety of microorganisms including Bacillus and Aspergillus, with most commercial amylases being produced from bacterial sources such as Bacillus licheniformis, Bacillus amyloliquefaciens, Bacillus subtilis, or Bacillus stearothermophilus. In recent years, the preferred enzymes in commercial use have been those from Bacillus licheniformis because of their heat stability and performance under commercial operating conditions.
In general, starch to fructose processing consists of four steps: liquefaction of granular starch, saccharification of the liquefied starch into dextrose, purification, and isomerization to fructose. The object of a starch liquefaction process is to convert a concentrated suspension of starch polymer granules into a solution of soluble shorter chain length dextrins of low viscosity. This step is essential for convenient handling with standard equipment and for efficient conversion to glucose or other sugars. To liquefy granular starch, it is necessary to gelatinize the granules by raising the temperature of the granular starch to over about 72.degree. C. The heating process instantaneously disrupts the insoluble starch granules to produce a water soluble starch solution. The solubilized starch solution is then liquefied by .alpha.-amylase (EC 3.2.1.1.).
A common enzymatic liquefaction process involves adjusting the pH of a granular starch slurry to between 6.0 and 6.5, the pH optimum of .alpha.-amylase derived from Bacillus licheniformis, with the addition of calcium hydroxide, sodium hydroxide or sodium carbonate. The addition of calcium hydroxide has the advantage of also providing calcium ions which are known to stabilize the .alpha.-amylases against inactivation. Upon addition of .alpha.-amylases, the suspension is pumped through a steam jet to instantaneously raise the temperature to between 80-115.degree. C. The starch is immediately gelatinized and, due to the presence of .alpha.-amylases, depolymerized through random hydrolysis of a (1-4) glycosidic bonds to a fluid mass which is easily pumped.
In a second variation to the liquefaction process, .alpha.-amylase is added to the starch suspension, the suspension is held at a temperature of 80-100.degree. C. to partially hydrolyze the starch granules, and the partially hydrolyzed starch suspension is pumped through a jet at temperatures in excess of about 105.degree. C. to thoroughly gelatinize any remaining granular structure. After cooling the gelatinized starch, a second addition of .alpha.-amylase can be made to further hydrolyze the starch.
A third variation of this process is called the dry milling process. In dry milling, whole grain is ground and combined with water. The germ is optionally removed by flotation separation or equivalent techniques. The resulting mixture, which contains starch, fiber, protein and other components of the grain, is liquefied using .alpha.-amylase. The general practice in the art is to undertake enzymatic liquefaction at a lower temperature when using the dry milling process. Generally, low temperature liquefaction is believed to be less efficient than high temperature liquefaction in converting starch to soluble dextrins.
Typically, after gelatinization the starch solution is held at an elevated temperature in the presence of .alpha.-amylase until a DE of 10-20 is achieved, usually a period of 1-3 hours. Dextrose equivalent (DE) is the industry standard for measuring the concentration of total reducing sugars, calculated as D-glucose on a dry weight basis. Unhydrolyzed granular starch has a DE of virtually zero, whereas the DE of D-glucose is defined as 100.
The maximum temperature at which the starch solution containing .alpha.-amylase can be held depends upon the microbial source from which the enzyme was obtained and the molecular structure of the .alpha.-amylase molecule. .alpha.-Amylases produced by wild type strains of Bacillus subtilis or Bacillus amyloliquefaciens are typically used at temperatures no greater than about 90.degree. C. due to excessively rapid thermal inactivation above that temperature, whereas .alpha.-amylases produced by wild type strains of Bacillus licheniformis can be used at temperatures up to about 110.degree. C. The presence of starch and calcium ion are known to stabilize .alpha.-amylases against inactivation. Nonetheless, .alpha.-amylases are used at pH values above 6 to protect against rapid inactivation. At low temperatures, .alpha.-amylase from Bacillus licheniformis is known to display hydrolyzing activity on starch substrate at pH values as low as 5. However, when the enzyme is used for starch hydrolysis at common jet temperatures, e.g., between 102.degree. C. and 109.degree. C., the pH must be maintained above at least pH 5.7 to avoid excessively rapid inactivation. The pH requirement unfortunately provides a narrow window of processing opportunity because pH values above 6.0 result in undesirable by-products, e.g., maltulose. Therefore, in reality, liquefaction pH is generally maintained between 5.9 and 6.0 to attain a satisfactory yield of hydrolyzed starch.
Another problem relating to pH of liquefaction is the need to raise the pH of the starch suspension from about 4, the pH of a corn starch suspension as it comes from the wet milling stage, to 5.9-6.0. This pH adjustment requires the costly addition of acid neutralizing chemicals and also requires additional ion-exchange refining of the final starch conversion product to remove the chemical. Moreover, the next process step after liquefaction, typically saccharification of the liquefied starch into glucose with glucoamylase, requires a pH of 4-4.5; therefore, the pH must be adjusted back down from 5.9-6.0 to 4-4.5; requiring additional chemical addition and refining steps.
Subsequent to liquefaction, the processed starch is saccharified to glucose with glucoamylase. A problem with present processes occurs when residual starch is present in the saccharification mixture due to an incomplete liquefaction of the starch, e.g., inefficient amylose hydrolysis by amylase. Residual starch is highly resistant to glucoamylase hydrolysis. It represents a yield loss and interferes with downstream filtration of the syrups.
Additionally, many .alpha.-amylases are known to require the addition of calcium ion for stability. This further increases the cost of liquefaction.
In U.S. Pat. No. 5,322,778, liquefaction between pH 4.0 and 6.0 was achieved by adding an antioxidant such as bisulfite or a salt thereof, ascorbic acid or a salt thereof, erythorbic acid, or phenolic antioxidants such as butylated hydroxyanisole, butylated hydroxytoluene, or a-tocopherol to the liquefaction slurry. According to this patent, sodium bisulfite must be added in a concentration of greater than 5 mM.
In U.S. Pat. No. 5,180,669, liquefaction between a pH of 5.0 to 6.0 was achieved by the addition of carbonate ion in excess of the amount needed to buffer the solution to the ground starch slurry. Due to an increased pH effect which occurs with addition of carbonate ion, the slurry is generally neutralized by adding a source of hydrogen ion, for example, an inorganic acid such as hydrochloric acid or sulfuric acid.
In PCT Publication No. WO 95/35382, a mutant .alpha.-amylase is described having improved oxidation stability and having changes at positions 104, 128, 187 and/or 188 in B. licheniformis .alpha.-amylase.
In PCT Publication No. WO 96/23873, mutant .alpha.-amylases are described which have any of a number of mutations.
In PCT Publication No. WO 94/02597, a mutant .alpha.-amylase having improved oxidative stability is described wherein one or more methionines are replaced by any amino acid except cysteine or methionine.
In PCT publication No. WO 94/18314, a mutant .alpha.-amylase having improved oxidative stability is described wherein one or more of the methionine, tryptophan, cysteine, histidine or tyrosine residues is replaced with a non-oxidizable amino acid.
In PCT Publication No. WO 91/00353, the performance characteristics and problems associated with liquefaction with wild type Bacillus licheniformis .alpha.-amylase are approached by genetically engineering the .alpha.-amylase to include the specific substitutions Ala-111-Thr, His-133-Tyr and/or Thr-149-IIe.
Studies using recombinant DNA techniques to explore which residues are important for the catalytic activity of amylases and/or to explore the effect of modifying certain amino acids within the active site of various amylases and glycosylases have been conducted by various researchers (Vihinen et al., J. Biochem., Vol. 107, pp. 267-272 (1990); Holm et al., Protein Engineering, Vol. 3, pp. 181-191 (1990); Takase et al., Biochemica et Biophysica Acta, Vol. 1120, pp. 281-288 (1992); Matsui et al., FEBS Letters, Vol. 310, pp. 216-218 (1992); Matsui et al., Biochemistry, Vol. 33, pp. 451-458 (1992); Sogaard et al., J. Biol. Chem., Vol. 268, pp. 22480-22484 (1993); Sogaard et al., Carbohydrate Polymers, Vol. 21, pp. 137-146 (1993); Svensson, Plant Mol. Biol., Vol. 25, pp. 141-157 (1994); Svensson et al., J. Biotech. Vol. 29, pp. 1-37 (1993)). Researchers have also studied which residues are important for thermal stability (Suzuki et al., J. Biol. Chem., Vol. 264, pp. 18933-18938 (1989); Watanabe et al., Eur. J. Biochem. Vol. 226, pp. 277-283 (1994)); and one group has used such methods to introduce mutations at various histidine residues in a Bacillus licheniformis amylase, the rationale being that Bacillus licheniformis amylase which is known to be relatively thermostable when compared to other similar Bacillus amylases, has an excess of histidines and, therefore, it was suggested that replacing a histidine could affect the thermostability of the enzyme. This work resulted in the identification of stabilizing mutations at the histidine residue at the +133 position and the alanine residue at position +209 (Declerck et al., J. Biol. Chem., Vol. 265, pp. 15481-15488 (1990); FR 2 665 178-A1; Joyet et al., Bio/Technology, Vol. 10, pp. 1579-1583 (1992)).
The introduction of di-sulphide bonds into proteins by site-directed mutagenesis affords a means of stabilizing native, folded conformations, see e.g., Villafranca et al., Science, Vol. 222, pp. 782-788 (1983). Hazes et al., Prot. Eng., Vol. 2, No. 2, pp. 119-125 (1988) suggest introducing disulfide bonds to a protein via a modeling algorithm which starts with the generation of the C.beta. position from the N, C.alpha. and C atom positions available from a known three-dimensional model. A first set of residues is selected on the basis of the C.beta.-C.beta. distances; the S.gamma. positions are generated which satisfy the requirement that, with ideal values for the C.alpha.-C.beta. and C.beta.-S.gamma. bond lengths and for the bond angle at C.beta., the distance between the S.gamma. of residue 1 and C.beta. of residue 2 in a pair (determined by the bond angle at S.gamma.2) is at, or very close to, its ideal value; and the two acceptable S.gamma. positions are found for each cysteine and the four different conformations for each disulfide bond established. Finally the four conformations are subjected to energy minimization procedure to remove large deviations from ideal geometry and their final energies calculated. Sowdhamini et al., Prot. Eng., Vol. 3, No. 2, pp. 95-103 (1989) discloses that the introduction of disulfide bonds into proteins by site directed mutagenesis affords a means of stabilizing native folded conformations and suggests computer modeling techniques for assessing the stereochemical suitability of pairs of residues in proteins as potential sites for introduction of cysteine disulfide crosslinks. The authors suggest that the elemental condition for considering residue positions in proteins, as potential sites for cysteine introduction, to generate unstrained disulfides is that the alpha carbon distance between the two cysteine residues to be joined via the disulfide bond (C.alpha.-C.alpha.) be less than or equal to 6.5 Angstroms and that the beta carbon distance between the two cysteine residues to be joined via the disulfide bond (C.beta.-C.beta.) distance of less than or equal to 4.5 Angstroms.
Despite the advances made in the prior art, a need exists for an .alpha.-amylase which is more effective in commercial liquefaction processes but which allows activity at lower pH than currently practical. Additionally, a need exists for improved amylases having characteristics which makes them more effective under the conditions of detergent use. Because commercially available amylases are not acceptable under many conditions due to stability problems, for example, the high alkalinity and oxidant (bleach) levels associated with detergents, or temperatures under which they operate, there is a need for an amylase having altered, and preferably increased, performance profiles under such conditions.